The long-term goal of this project is to understand how alternative splicing is regulated in the mammalian central nervous system (CNS). Studies carried out during the previous funding period established the Hu family of paraneoplastic neurologic disease (PND) antigens in neurons as alternative splicing regulators. More recently, preliminary data from a yeast two-hybrid screen uncovered a potential role for Hu proteins as mediators that link transcription with splicing. Specifically, HuC interacts with histone H3 and histone deacetylase HDAC2. Importantly, Hu proteins associate with RNAPII engaged in elongation and expression of Hu proteins correlates with higher level of acetylated H3 and H4 in an internal region of the Neurofibromatosis Type 1 (NF1) gene surrounding the alternatively spliced exon 23a. The central goal of this proposal is to test the hypothesis that Hu proteins regulate splicing in a co-transcriptional manner by directly interacting with chromatin bound histone H3 and/or the chromatin remodeling factor HDAC2. To define the molecular basis of the mechanisms through which these interactions regulate pre-mRNA splicing in CNS neurons, three specific aims will be pursued. In aim I, a robust mouse system will be established for the derivation of homogeneous CNS neurons from mouse ES cells to study neuron-specific alternative splicing. This system will allow us to combine genetic and biochemical approaches to investigate splicing regulation in neuronal cells. In aim II, the potential involvement of the transcription machinery in Hu-mediated regulation of alternative splicing in neurons will be examined. The specificity of the Hu-HDAC interaction will be determined and deletion and point mutational analysis will be carried out to define the nature of these interactions. Further studies will test whether Hu proteins are associated with the promoter region of NF1 and other genes in neurons. In aim III, the functional consequences of the Hu-HDAC/H3 interactions in Hu-mediated alternative splicing in neurons will be determined. Using exon 23a of the NF1 pre-mRNA as the substrate, the effect of transcription elongation rate on alternative splicing will be examined. These studies will provide fundamental insights into the mechanisms that control tissue-specific, particularly neuron-specific, alternative RNA splicing and coupling of transcription and splicing. The CNS neuronal differentiation system to be developed will serve as a valuable new alternative model in studies of neuron-specific splicing regulation.